Immunity
Article Late Developmental Plasticity in the T Helper 17 Lineage Yun Kyung Lee,1,2 Henrietta Turner,1 Craig L. Maynard,1 James R. Oliver,1 Dongquan Chen,3 Charles O. Elson,3 and Casey T. Weaver1,* 1Department
of Pathology of Microbiology 3Department of Medicine University of Alabama at Birmingham, Birmingham, AL 35294, USA *Correspondence:
[email protected] DOI 10.1016/j.immuni.2008.11.005 2Department
SUMMARY
Development of T helper (Th) 17 cells requires transforming growth factor (TGF)-b and interleukin (IL)-6 and is independent of the Th1 pathway. Although T cells that produce interferon (IFN)-g are a recognized feature of Th17 cell responses, mice deficient for STAT4 and T-bet—two prototypical Th1 transcription factors—are protected from autoimmunity associated with Th17 pathogenesis. To examine the fate and pathogenic potential of Th17 cells and origin of IFN-g-producing T cells that emerge during Th17 immunity, we developed IL-17F reporter mice that identify cells committed to expression of IL-17F and IL-17A. Th17 cells required TGF-b for sustained expression of IL-17F and IL-17A. In the absence of TGF-b, both IL-23 and IL-12 acted to suppress IL-17 and enhance IFN-g production in a STAT4- and T-bet-dependent manner, albeit with distinct efficiencies. These results support a model of late Th17 developmental plasticity with implications for autoimmunity and host defense. INTRODUCTION T helper 17 is a third major CD4+ T cell effector subset important in host protection and autoinflammatory disorders (McGeachy and Cua, 2008; Stockinger and Veldhoen, 2007; Weaver et al., 2007). Th17 cells are characterized by production of the cytokines, interleukin (IL)-17A, and IL-17F. Although originally proposed as a branch of the Th1 lineage (Bettelli and Kuchroo, 2005; McKenzie et al., 2006), IL-17-producing effectors are now thought to develop independently of both Th1 and Th2 lineages (Harrington et al., 2005; Park et al., 2005). Th17 cells differentiate from naive CD4+ T cell precursors in response to transforming growth factor (TGF)-b and IL-6; the Th1 lineage-associated factors signal transducer and activator of transcription (STAT)1, T-bet, and STAT4, as well as the Th2 factors STAT6 and GATA3, are dispensable for Th17 differentiation (Bettelli et al., 2006; Harrington et al., 2005; Mangan et al., 2006; Park et al., 2005; Veldhoen et al., 2006). Th17 development is inhibited by the Th1 cytokine and interferon (IFN)-g, as well other cytokines that activate STAT1 92 Immunity 30, 92–107, January 16, 2009 ª2009 Elsevier Inc.
(e.g., type I interferons and IL-27), by the Th2 cytokine IL-4 (Batten et al., 2006; Harrington et al., 2005; Park et al., 2005; Villarino et al., 2003) and is constrained by IL-2 signaling via STAT5 (Laurence et al., 2007). TGF-b, which is required for Th17 development, has suppressive effects on Th1 development (Gorham et al., 1998), through inhibitory effects on STAT4 and T-bet signaling, and also inhibits Th2 development (Gorelik et al., 2000; Lin et al., 2005). STAT3 plays a central role in Th17 development, coupling early IL-6 signaling to induction of IL-21, an autocrine factor implicated in the progression of Th17 development (Harris et al., 2007; Korn et al., 2007; Mathur et al., 2007; Nurieva et al., 2007; Yang et al., 2007; Zhou et al., 2007). IL-21, also acting in part through STAT3 signaling, can induce expression of the transcription factors RORgt and RORa, which appear to be essential for Th17-lineage specification (Ivanov et al., 2006; Yang et al., 2008). Despite the developmental signals that distinguish Th17 and Th1 differentiation, there are interesting parallels between the two developmental programs. IL-23R is induced in developing Th17 cells and IL-12Rb2 is induced in developing Th1 cells to pair with the constitutively expressed IL-12Rb1 chain to confer responsiveness to IL-23 and IL-12, respectively. The IL-12Rb1 chain binds a common subunit of the IL-23 and IL-12 heterodimers—IL-12p40 (or IL-12b)—that pairs with IL-23p19 (or IL-23a) or IL-12p35 (or IL-12a), respectively (Aggarwal et al., 2003; Oppmann et al., 2000; Parham et al., 2002). Activation of the IL-23 or IL-12 receptors drives late events downstream of early Th17 or Th1 lineage commitment (Harrington et al., 2005; Mangan et al., 2006). Whereas the IL-12 receptor potently activates STAT4 in Th1 cells, the IL-23 receptor predominantly activates STAT3, but also recruits some STAT4 (Mathur et al., 2007; Oppmann et al., 2000; Parham et al., 2002; Yang et al., 2007). The long-held association of autoimmune models such as experimental autoimmune encephalomyelitis (EAE), collageninduced arthritis (CIA), and inflammatory bowel disease (IBD) with Th1-mediated pathogenesis has been revised in view of the requirement for IL-23, but not IL-12, for disease in these models (Cua et al., 2003; Langrish et al., 2005; Murphy et al., 2003). This has given rise to a new Th17-mediated pathogenesis paradigm (McGeachy and Cua, 2008). However, early reports that linked IL-23 to induction of IL-17 expression by memory CD4+ T cells also demonstrated induction of IFN-g expression (Oppmann et al., 2000), and IFN-g-producing T cells are typically found in association with IL-17-producing T cells in the context
Immunity Th17 Developmental Plasticity
of both infectious and autoimmune inflammation. Interestingly, whereas mice lacking key components of the Th1 pathway (e.g., IL-12p35, IFN-g, IFNgR, IL-12Rb2, and STAT1) retain susceptibility to autoimmunity (Becher et al., 2002; Bettelli et al., 2004; Ferber et al., 1996; Gran et al., 2002; Willenborg et al., 1996; Zhang et al., 2003), deficiency of STAT4 and T-bet can preclude disease (Bettelli et al., 2004; Chitnis et al., 2001; Neurath et al., 2002), suggesting either that STAT4 and T-bet have important roles in both Th1 and Th17 inflammation or that cooperation of Th1 and Th17 responses is necessary for immune pathogenesis in certain settings. Although IL-23 is dispensable for Th17 commitment (Mangan et al., 2006; Veldhoen et al., 2006), its requirement for Th17associated immune protection or autoimmunity is undisputed, and it is speculated to enhance and/or maintain the Th17 phenotype (Veldhoen et al., 2006; Yang et al., 2007; Zhou et al., 2007). Indeed, Th17 cells differentiated by TGF-b and IL-6 in the absence of IL-23 have been shown to have a protective rather than pathogenic role in a model of EAE (McGeachy et al., 2007), consistent with an essential role for IL-23 in Th17 effector function. Nevertheless, the precise effects of IL-23 on Th17 lineage stability remain unclear (McGeachy and Cua, 2008; Weaver et al., 2007). To explore the role of IL-23 in late Th17 development, we have examined the fate and phenotype of Th17 cells generated and maintained over the long term under defined cytokine conditions. Using an IL-17F reporter mouse to identify developing Th17 cells committed to expression of both IL-17F and IL-17A, we found that TGF-b was essential for maintenance of IL-17 expression, whereas IL-23 was not. Propagation of committed Th17 precursors in the presence of IL-23 without TGF-b resulted in progressive extinction of IL-17F and, to a lesser extent, IL-17A and promoted the emergence of IFN-g-producing cells that lacked IL-17 expression. Remarkably, Th17 cells maintained over the long term by TGF-b demonstrated persistent responsiveness to IL-12 despite relatively low expression of IL-12Rb2. Stimulation of Th17 cells with IL-12 induced a rapid, STAT4and T-bet-dependent transition marked by extinction of RORgt, RORa, IL-17A, and IL-17F and an induction of a Th1-like geneexpression signature. Th17-committed cells generated in the presence or absence of IL-23 induced rapid development of colitis when transferred into immunodeficient recipients and showed transition of a subset of cells to IFN-g expression. Blockade of IL-23 ablated development of colitis, but did not prevent persistence of IL-17-expressing cells nor transition to a subset of IFN-g producers, whereas blockade of IL-23 and IL-12 ablated development of colitis and markedly diminished persistence of transferred Th17 cells. These findings support substantial developmental plasticity of the Th17 lineage and identify a mechanism for latent Th1-like responsiveness of Th17 cells that provides a basis for understanding the relationship between Th17 and Th1 mediated immunity. RESULTS TGF-b Is Required for Maintenance of IL-17 Expression by Th17 Cells Although IL-23 is required for optimal expression of IL-17 under conditions of limiting TGF-b during Th17 development (Zhou
et al., 2007), it is dispensable for Th17 commitment and its role in late Th17 development remains incompletely defined. To examine the possible requirement for IL-23 in maintenance of the Th17 phenotype, we propagated Th17 cells generated in the absence of IL-23 under defined cytokine conditions in vitro (Figure 1). Th17 cells maintained in the continued presence of exogenous TGF-b demonstrated an increase in the frequency of IL-17A+ progeny that was comparable in the presence or absence of exogenous IL-6 or IL-23. Neutralization of possible endogenous IL-6 had no detectable effect on the maintenance of IL-17 expression, consistent with its being nonessential for late events in Th17 development sustained by IL-21 (Korn et al., 2007; Nurieva et al., 2007; Zhou et al., 2007, and data not shown). In all cultures maintained with TGF-b, a subset of IL-17A+ cells coexpressed IFN-g, despite the absence of IL-12 and irrespective of the addition (or absence) of IL-23 or IL-6. Notably, whereas the frequency of IL-17A+ cells decreased under conditions of IL-23 stimulation in the absence of TGF-b, the frequency of cells that expressed IFN-g, without coexpression of IL-17A, markedly increased. Because a minor fraction of IFN-g+ cells was generated under Th17-polarizing conditions in the absence of IL-12 and IL-23 (Figure 1A), we postulated that IL-23 might act on this small subset as a source for outgrowth of IFN-g+ progeny under conditions in which TGF-b was deficient. To address this, we used naive CD4+ T cells from IFN-g reporter mice (IfngThy1.1/Thy1.1) that permit specific depletion of IFN-g-producing cells via the Thy1.1 reporter molecule (Harrington et al., 2008). Th17 cells were polarized as before, except that the small fraction of IFN-g+ (Thy1.1+) cells was depleted prior to further propagation with either TGF-b and IL-6 or IL-23 (Figure 1B). Similar to previous results, the fraction of IFN-g-producing cells was significantly increased by IL-23 stimulation in the absence of continued TGF-b, whereas IL-17A-producing cells were comparably amplified in the presence of TGF-b—as were the subset of IL-17A+IFNg+ cells. Collectively, these results suggest that TGF-b is a critical factor for both differentiation and maintenance of IL-17producing effector cells; Th17 cells do not stably express IL-17A in the absence of continuous TGF-b signaling. In contrast, IL-23 appears to be dispensable for both the induction and maintenance of Th17 cells, but instead is associated with transition to progeny that extinguishes IL-17 expression and enhances IFN-g expression under conditions of low or absent TGF-b. Thus, developing Th17 cells can give rise to ‘‘Th1-like’’ progeny in the absence of ongoing TGF-b signaling. Importantly, the converse is not true; Th1-polarized cells are resistant to transition to a Th17 cytokine-expression pattern when propagated under Th17 conditions (Figure S1 available online and Harrington et al., 2005). Accordingly, Th17 cells appear to have greater plasticity with respect to their cytokine expression during late stages of development. Th17 Cells Respond to IL-12 to Rapidly Extinguish IL-17 and Promote IFN-g Expression Th17 cells upregulate IL-23R as part of their developmental program to confer responsiveness to IL-23 (Harrington et al., 2005; Mangan et al., 2006; Oppmann et al., 2000; Zhou et al., 2007). In contrast, developing Th1 cells upregulate IL-12Rb2 to confer IL-12 responsiveness, and it is thought that the reciprocal Immunity 30, 92–107, January 16, 2009 ª2009 Elsevier Inc. 93
Immunity Th17 Developmental Plasticity
Figure 1. Requirement for TGF-b in the Maintenance of IL-17 Expression by Th17-Polarized Cells (A) FACS-sorted naive CD4+ T cells from OT-II TCR transgenic mice were cultured with irradiated Il12b/ (IL-12p40-deficient) splenic feeder cells and 5 mg/ml OVAp for 7 days under Th17-polarizing conditions (TGF-b, 5 ng/ml; IL-6, 20 ng/ml; anti-IFN-g, 10 mg/ml; anti-IL-4, 10 mg/ml), then stained intracellularly for IL-17A and IFN-g after PMA/ionomycin activation for 5 hr in the presence of monensin (1 culture; left panel). Cells were harvested and restimulated with irradiated Il12b/ splenic feeder cells in the presence of indicated cytokine(s) at the same doses or, in the case of IL-23 and IL-12, at 1 ng/ml, with anti-IFN-g, antiIL-4, and OVAp for an additional two rounds (7 days each) and stained intracelluarly for IL-17A and IFN-g after PMA-ionomycin activation (post 3 culture; right panels). (B) IfngThy1.1/Thy1.1 OT-II naive CD4+ T cells were cultured with irradiated Il12b/ splenic feeder cells under Th17-polarizing conditions for 6 days and a fraction of the recovered cells were stained intracellularly for IL-17A and IFN-g after PMAionomycin activation (left panel). A second fraction of recovered CD4+ T cells were activated for 5 hr with OVAp and Il12b/ feeder cells, and Thy1.1+ (IFN-g+) cells were depleted by magnetic sorting. Thy1.1 depletion was confirmed by surface staining for Thy1.1 (middle panel), and the isolated Thy1.1- cells were cultured with Il12b/ feeder cells and OVAp and either TGF-b plus IL-6 or IL-23 in the presence of anti-IFNg plus anti-IL-4 for three additional rounds (right panels), then stained intracellulary for IL-17A and IFN-g after PMA-ionomycin activation. Numbers in each quadrant indicate percentages of total CD4+ T cells. All data are representative of at least three independent experiments.
expression of IL-23R and IL-12Rb2, both of which pair with constitutively expressed IL-12Rb1, is central to differential development of Th17 and Th1 cells, respectively (Harrington et al., 2005; Mangan et al., 2006; Zhou et al., 2007). Given the observed deviation of Th17 cells toward enhanced IFN-g expression in the presence of IL-23, we examined the possible effect of IL-12 on Th17 cells (Figure 2). Naive CD4+ T cells polarized with Th17inductive cytokines and antigen-presenting cells (APCs) deficient in expression of IL-12 and IL-23 (Il12b/) were divided into fractions that were maintained for one to three more cycles of stimulation with addition of the indicated exogenous cytokines. Consistent with previous results, TGF-b was required to maintain high frequencies of IL-17-expressing cells. Even in the absence of added cytokines, antigen stimulation alone (OVAp) induced progressive decline in IL-17A expression and enhanced IFN-g 94 Immunity 30, 92–107, January 16, 2009 ª2009 Elsevier Inc.
expression in the absence of exogenous TGF-b. IL-23 signaling in the absence of TGF-b significantly and consistently increased the frequency of IFN-g+ cells and decreased IL-17A+ cells relative to the absence of TGF-b alone. Remarkably, a large majority of Th17 cells restimulated with IL-12 in the absence of TGF-b produced IFN-g (72%) and IL17A expression was markedly extinguished, such that a small minority of cells produced IL-17A (8%) after only a single round of culture. With continued maintenance in IL-12, most cells became IFN-g single-producers (85%) and IL-17A was nearly completely extinguished (
